Stem cell genetics

The Stem cell genetics group focuses on developing novel genetic engineering technologies in embryonic stem cells (ES)
and induced pluripotent stem (iPS) cells that can then be used to carry out genetic screening and may produce new
healthcare therapies.

Pluripotent stem cells - cells that have the ability to become almost any kind of mature cell - are valuable tools for
improving human health in two important ways:

they enable in-depth investigation of the genetic basis of disease

they have great potential to provide new genetic therapies

Mouse embryonic stem (ES) cells are one type of pluripotent stem cells that have been used extensively over the past
three decades to discover more about disease. Using genetic engineering techniques to knock out each gene in the mouse
genome, mouse ES cells have allowed scientists to study the function of mammalian genes both in cultured cells and in
the mouse itself.

Human ES cells, and the emerging field of induced pluripotent stem (iPS) cells, hold great promise in regenerative
medicine and in studying the mechanics of human disease. To realise and maximise the potential of these stem cells for
research and therapy, we need to create robust, efficient and precise genetic engineering techniques.

Our aims are to develop reliable and scalable genetic engineering technologies and to apply them to genetic screening
and new healthcare therapies using ES and iPS cells.

Background

Embryonic stem cells (ES) are a useful research tool because they can multiply indefinitely while maintaining their
ability to develop into any type of cell found in the body. Furthermore, their genomes can be modified in a targeted
way using homologous recombination. For the past three decades mouse ES cells have been successfully used to study
loss-of-function effects in vivo as well as in vitro. However, the diploid nature of their genome
means that both sets of each gene must be inactivated for successful phenotype-driven forward genetic screens, which
has hampered research efforts.

Human ES cells are a relatively newer cell type with great research potential, but technical difficulties have
hindered their use. These cells were first reported in 1998 and have considerably different properties from mouse ES
cells, although both cell types are established from pre-implantation embryos. Compared with mouse ES cells, human ES
cells are morphologically different, more prone to cell death upon single cell dissociation, and use a different
signalling pathway to maintain their pluripotency. Moreover, homologous recombination is less efficient in human ES
cells than in mouse ES cells.

However, the new technique of directly reprogramming of human somatic cells into human induced pluripotent stem (iPS)
cells overcomes many the problems encountered with human ES cells. This approach is opening the door to cell-based
genetic therapies, highlighting the need to develop robust and efficient genetic engineering techniques in these iPS
cells.

Research

Our aims

The group's main focus is to develop novel genetic engineering technologies in human and mouse pluripotent stem cells
and to apply them to genetic screening and new healthcare therapies.

Recessive genetic screening in mouse ES cells

We developed an efficient method to conduct recessive genetic screens in mouse ES cells by using the
hyper-recombination phenotype of Bloom helicase-deficiency to produce genome-wide homozygous mutations. Using this
technique, we identified novel factors involved in the toxification of Aeromonas toxin, aerolysin.

We are now expanding our recessive genetic screens by using newly isolated mouse haploid ES cells. These cells
contain only one set of the genome and therefore can be altered by a 'single hit'. Because of this, these ES cells
are a genetic tool of unprecedented potential for gene function studies of mammalian biological systems.

High-throughput sequencing will enable us able to identify mutations introduced into mouse haploid ES cells, allowing
us to conduct a wide range of genetic screens: from finding the genes required for ES cell differentiation to looking
for those involved in viral replication.

The piggyBac transposon system

piggyBac is a moth-derived DNA transposon (jumping gene) and is one of the most active transposon systems in
mammalian cells. Transposons have been used in a wide range of organisms for genetic studies - especially random
mutagenesis. The transposon sequence can be used as a molecular tag in the DNA and, therefore, we can easily identify
mutated genes by searching for transposon integration sites.

In addition, the piggyBac transposon has a unique property that offers us a powerful way to reprogramme iPS
cells in a manner that could be used in treatments. piggyBac has traceless excision, which enables removal
of the transposon from the genome without altering the cells' DNA sequences. Using this important property, we have
established a transposon-based, factor-free, method for iPS cell reprogramming. The iPS cells established by this
means are clean from potentially mutagenic changes.

We are also developing more active transposon systems, to expand the usefulness of this approach in mammalian genetic
research and gene therapy. In collaboration with Professor Nancy Craig at Johns Hopkins University, we have developed
a hyperactive piggyBac transposase, which can increase transposition efficiency by one order of magnitude.

Human iPS cells hold great promise for autologous cell-based therapy in a broad range of disorders. However, creating
human iPS cells from a person with an inherited disease to treat their condition requires not only the correction of
the disease-causing mutation, but also for it to be done cleanly to avoid other DNA changes or unwanted side-effects.

Until recently, cleanly editing the genome of human iPS cells has been particularly challenging because of the lack
of necessary technologies. But now we have developed a technique that combines using zinc-finger nuclease (ZFN) with
the hyperactive piggyBac transposon system to achieve efficient and precise genome editing. Using this
approach, we have successfully performed genetic correction of a major inherited metabolic disease - alpha-1
antitrypsin deficiency - in human iPS cells.

To check that unwanted mutations were not introduced during the 'genetic surgery', we conducted high-resolution
analysis of the corrected genome using array-based comparative genomic hybridization, single nucleotide polymorphism
array and exome-sequencing. This revealed that genomic integrity of iPS cells is maintained throughout genetic
correction.

This approach is now even more accessible with novel nuclease technologies such as TALE nuclease and the Cas9
systems. Patient-derived iPS cells and their disease-corrected counterparts offer the opportunity to carry out the
best controlled in vitro modelling of diseases. We will investigate the potential of genome editing in human
iPS cells to develop new ways to investigate the molecular mechanisms underlying disease as well as create new
cell-based therapies.

Collaborations

On campus, we work closely with the following Sanger Institute faculty members: